Adsorbent for chromatographic purification of virus



United States Patent Oifice 3,509,070 Patented Apr. 28, 1970 3,509,070ADSORBENT FOR CHROMATOGRAPHIC PURIFICATION OF VIRUS Milton Lapidus,Rosemont, Pa., assignor to American Home Products Corporation, New York,N.Y., a corporation of Delaware No Drawing. Continuation-in-part ofapplication Ser. No. 497,540, Oct. 18, 1965, which is acontinuation-in-part of application Ser. No. 396,726, Sept. 15, 1964.ThlS application June 13, 1968, Ser. No. 736,561

Int. Cl. B01j 11/82; C01b 25/32; A61k 23/00 U.S. Cl. 252-437 7 ClaimsABSTRACT OF THE DISCLOSURE This invention relates to an alkali treatedcalcium dihydrogen orthophosphate monohydrate adsorbent. Further, itconcerns an alkali and trimetaphosphate treated calcium dihydrogenorthophosphate monohydrate adsorbent. Still further, it relates to theprocesses for the preparation of these adsorbents and to their use invirus purification.

This application is a continuation-in-part of co-pending US.application, Ser. No. 497,540, filed Oct. 18, 1965 and now abandonedwhich is a continuation-in-part of its parent US. patent application,Ser. No. 396,726, filed on Sept. 15, 1964, which is now abandoned.

This invention relates to the purification and concentration of virus.More particularly, this invention relates to new and improved adsorbentcompositions particularly effective in purifying influenza virus. Thepresent invention also encompasses the novel method by which these newadsorbent compositions are prepared as well as the method by which thesame can be utilized in preparing highly purified forms of influenzavirus.

In the US. Patent No. 3,368,867, entitled, Chromato graphic Purificationof Influenza Virus With Brushite Modified by Autoclaving, filed on Sept.15, 1964 and issued on Feb. 13, 1968, there is described a new adsorbentof the calcium phosphate family, namely, autoclaved brushite. Asdescribed therein, it was found quite unexpectedly that by autoclavingbrushite, separation and concentration of influenza virus from allantoicfluids containing the same could be substantially improved. It isfurther shown in US. Patent No. 3,368,867 that the autoclaved brushitemade possible a substantial improvement in the degree or level ofpurification of Sharples concentrated B strain influenza virus.

It has now been discovered according to the present invention thatanother calcium salt, namely, calcium dihydrogen orthophosphatemonohydrate [Ca(H PO.,)- H O] if treated according to the method of thepresent invention is eflective in separating various strains of virusincluding the B strain from influenza infected allantoic fluid. It hasalso been found that the effectiveness of this adsorbent composition isfurther improved if the adsorbent is treated with an alkali metaltrimetaphosphate e.g. sodium trimetaphosphate or potassiumtrimetaphosphate prior to use. Utilization of an alkali metaltrimetaphosphate for this purpose as distinguished from the conventionalphosphate buffers such as dibasic sodium phosphate (Na HPO affords anunexpected increase in recovery of A strains of influenza virus and anincreased purification factor, generally in the order of four to fivetimes.

In addition and more importantly, the new adsorbent of the presentinvention when treated with an alkali metal trimetaphosphatesubstantially improves both the yield and purification of B strains ofinfluenza virus from the fluids in which such strains are normallypresent. This improvement is in the order of a four to five foldincrease in recovery and up to a ten fold increase in the degree orlevel of the virus purification obtained. This and other advantages areprovided by the novel adsorbent of the present invention and the methodin which the same is used.

Considered in its broadest aspect, the new adsorbent of the presentinvention is the monohydrate of calcium dihydrogen orthophosphate whichis treated with an aqueous solution of an alkali to provide a suspensionhaving a pH in the range of about 5.5 to about 8.0, preferably about6.5. As employed herein the term alkali is meant to comprise thoseaqueous solutions which contain compounds which yield hydroxyl ions upondissolution. Many such alkali will readily suggest themselves to thoseskilled in the chemical art. Some examples thereof are: sodium orpotassium nitrate, sodium or potassium acetate and sodium or potassiumhydroxide. In a preferred embodiment of this invention, calciumdihydrogen orthophosphate monohydrate be mixed with a limited quarttityof water such as from about one to not more than about five parts ofwater per part of salt and preferably from about three to about fiveparts of water per part of salt should be used. After addition of themonohydrate of calcium dihydrogen orthophosphate, stirring of theresulting suspension is continued during which the alkali is addedslowly over a period of from about onehalf to about two hours until theadjusted pH of about 5.5 to about 8.0 is achieved, preferably about 6.5.During this period the reaction mixture is kept at a temperature of notmore than about 40 C. and preferably a temperature of from about 20 to30 C., although temperatures as low as about 0 C. may be employed.Thereafter, the alkali treated adsorbent is permitted to settle and thesupernatant liquid is decanted. The residue is then washed with wateruntil the pH of the water Wash is at least 0.2 unit higher than theadjusted pH of the above suspension and is in the range of from about5.7 to about 8.2. The alkali treated calcium dihydrogen orth- Ophosphatemonohydrate adsorbent may then be kept in the supernatant fluid or thelatter can be decanted to leave the solid adsorbent in a stable formWhich is ready for use. When the alali treated adsorbent is used forvirus purification, the procedure employed is similar to thathereinafter described for the alkali and TMP treated adsorbent exceptthat the adsorbent is not treated with an alkali metal trimetaphosphate.Further, the virus containing fluid may or may not be TMP treated.

As previously indicated, the preparation of the alkali treated calciumdihydrogen orthophosphate monohydrate adsorbent according to the methoddescribed above provides an unexpectedly effective means for recoveringa highly purified virus from influenza infected source materials.However, it is an added feature of the present invention to increase andfurther activate this alkali treated adsorbent by treating the same withsufficient amount of an alkali metal trimetaphosphate (TMP) preferablysodium trimetaphosphate (Na P O until the pH of the adsorbent reached arange from about 7.5 to about 8.5, preferably about 8.0. At this pH, thealkali and TMP 3 I treated adsorbent is particularly effective, as isdemonstrated more clearly hereinafter, in separating influenza visusfrom allantoic fluid. This alkali and TMP adsorbent is particularlyeffective in recovering the B/GL strain of influenza virus fluidscontaining the same.

When the alkali and TMP treated calcium dihydrogen orthophosphatemonohydrate adsorbent is used for virus purification, it is placed orpacked in a conventional chromatographic column having the necessaryadsorbent support. If the pH of the adsorbent has not previously beenadjusted to the desired pH, e.g. a pH of about 8.0, the same is washedwith an aqueous alkali metal trimetaphosphate solution, e.g. sodiumtrimetaphosphate preferably at a molar content of about 0.01. Thevirus-containing fluid such: as allantoic fluid is usually diluted fromabout a 2:1 to about a 5:1 ratio with water and adjust to a pH range ofabout 7.0 to about 9.0 after the addition of the alkalimetaltrimetaphosphate. While effective purification of the virus can beobtained at these dilution and pH ranges, it has been found that higherflow rates are obtained in the column at a preferred dilution ratio ofabout 2:1 and a pH of about 8.

To concentrate the influenza virus, the alkali metal trimetaphosphatetreated and pH-adjusted allantoic fluid containing the virus is thenpoured onto and through the prepared column. The amount of virussolution delivered to the column will depend on the size of the columnand the quantity of adsorbent contained therein. It is preferred thatthe column be operated in a continuous manner and not permitted to rundry. When the total amount of infected allantoic fluid has been placedon the column, the virus is washed with an aqueous alkali metaltrimetaphosphate solution (pH 8), e.g. sodium trimetaphosphate.Thereafter, the virus is recovered by elution. In carrying out thiselution to provide a purified concentrate of the virus a conventionalbuffer with a pH in the range of about 5.0 to about 9.0 having a molarconcentration of about 0.5 M to about 2.0 M is employed. Preferably a1.0 M phosphate buffer e.g. dibasic sodium phosphate having a pH ofabout 6.5 to about 7.0 is used.

Reference now to the specific examples which follow will provide abetter understanding of the nature and characteristics of the newadsorbent composition of the present invention, the method by which thesame is prepared and the manner in which it is used.

EXAMPLE I Fifty grams of calcium dihydrogen orthophosphate monohydrate[Ca(H PO -H O] and 200 ml. of distilled water are stirred vigorously forfifteen minutes. With stirring a cold (4 C.) 3 N sodium hydroxidesolution is added dropwise thereto at room temperature over a period ofone-half hour until a pH of 6.5 is obtained. Thereafter, stirring iscontinued for one hour, the adsorbent is allowed to settle and thesupernatant is removed by dccantation. The adsorbent is washed with oneliter portions of distilled water until the supernatant liquid has a pHof 7.2-7.4. pH history of absorbent during the alkali treatment cycleConditions: pH

50 g. Ca(H PO -H O+2O0 ml. water 2.3

109 ml. 3 N sodium hydroxide 6.5

One hour stirring 6.5

1 liter water wash 6.8

1 liter water wash 7.05

1 liter water wash 7.3

EXAMPLE II One hundred grams of calcium dihydrogen orthophosphatemonohydrate [Ca(H PO -H O] and 500 ml. of distilled water are stirredfor a half hour. Thereafter, with continuous stirring at roomtemperature 2 N potassium hydroxide is added dropwise over a period oftwo hours until a pH of 6.5 is obtained. The stirring is continued foranother hour, then the supernatant liquid is decanted and the solidresidue Washed with two liter portions of distilled water until the pHof the supernatant liquid is 7.0. Thereafter, the supernatant liquid isdecanted to afford the stable adsorbent.

In like manner, a 1:3 ratio of calcium dihydrogen orthophosphatemonohyclrate and water is treated with a potassium bicarbonate solutionat 40 C. until a pH of 6.0 is obtained. Thereafter, the solid isseparated from the suspension and washed with water until a pH of 7.5 isachieved.

EXAMPLE III Repeating the procedure of Examples I and II a mixturecontaining 1:2 ratio of calcium dihydrogen orthophosphate monohydrateand water is treated with an aqueous sodium bicarbonate solution at 5 C.until the pH of the mixture is 5.5. Thereafter, the supernatant liquidis removed and the residue washed three times with two liter portions ofwater until the pH is in the range of 5.7 to 8.2.

-The above procedure is repeated with similar results using thefollowing alkali agents: sodium carbonate, potassium carbonate, sodiumnitrate, potassium nitrate, sodium acetate and potassium acetate.

EXAMPLE IV Fifty grams of calcium dihydrogen orthophosphate monohydrateand 200 ml. of distilled Water are stirred vigorously for fifteenminutes. Under conditions of continuous mixing, a cold (4 C.) 3 N sodiumhydroxide solution is added dropwise over a period of one-half houruntil a pH of 6.5 is reached. The contents ofthe reaction mixture iskept at 20-23" C. After stirring for one. hour, the adsorbent ispermitted to settle and the supernatantliquid is decanted. The adsorbentis Washed batchwise with one liter portions of distilled water until thepH of the supernatant liquid is 7.2-7.4. The adsorbent is then batchwashed with a 0.01 M sodium trimetaphosphate (pH 8) solution until a pHof 8 is reached.

pH history of adsorbent during alkali and TMP treatment cycleConditions: pH 50 g. Ca(H PO 'H 0+200 ml. water 2.3 109 ml. 3 N sodiumhydroxide (4 C.) 6.5 One hour stirring 6.5 1 liter water wash 6.8 1liter water wash 7.05 1 liter water wash 7.3 1 liter .01 M, pH 8, sodiumtrimetaphosphate wash 7.65 1 liter .01 M, pH 8, sodium trimetaphosphatewash 8.00

Similarly, with like results the above procedure is conducted with a0.01 M potassium trimetaphosphate solution.

EXAMPLE V The alkali treated adsorbents of Examples II and III arewashed with an aqueous solution of an alkali metal trimetaphosphate asdemonstrated in Example IV until the pH of the adsorbent is in the rangeof about 7.5 to about 8.5.

EXAMPLE v1 Fifty grams of Ca(H PO -H 0 treated according to Example IVis placed in a glass column containing a sintered glass disk to supportthe bed of 4.4 x 6 cm. size. To the column there is delivered fiveliters of diluted infected allantoic fluid containing B/GL virus. Thefluid having an adjusted pH of 9.0 contained 0.01 M sodiumtrimetaphosphate flow through the column over a period of two hours. Thecolumn is then washed with 200 ml. of 0.01 M sodium trimetaphosphate (pH8) solution and the virus eluted with 0.5 M phosphate buffer (pH 6.8)

solution. In the data reported below, column fraction 2 By the aboveprocedure, recovery of various strains of represents the recoveredvirus. influenza virus varied from 14 percent to 46.7 at purifica- LowryVolume, protein, CCA] Percent Sample ml. AA mg. mg. L.l P.F. recoveryInfected allantoic fluid 5,000 19, 000 925 20. Column fraction 1 5, 200954 Column fraction 2 100 12, 800 15 853. 3 41. 0 07 NoTE.CCA meansChick-Cell Agglutination Units and P.F. means Purification factor.

By the above procedure a recovery of 67 percent of 10 tion factors offrom 4.8 to 20.1. As will be noted, particthe B strain is obtained at apurification factor of 41.6. ularly with respect to the recovery of theB strain Great Similar results are afforded by the alkali and TMP Lakes(B/GL) a recovery of fourteen percent is attained treated adsorbentsdescribed in Example V When the at a purification factor of 4.8 withthis alkali treated-advirus-containing fluid is diluted from about a 2:1to sorbent which contrasts with the results of Example VI about a 5:1ratio with water, adjusted to a pH range in which the alkali and TMPtreated adsorbent affords a of about 7.0 to about 9.0 and eluted fromthe column recovery of 67 percent with a purification factor of 41.6.with a 0.3 M to a 2.0 M phosphate buffer having a pH In a similar mannerthe results obtained when purifying of about 5.0 to about 9.0. Astrain/Jap. 305 without an alkali metal trimetaphos- EXAMPLE VII phatetreatment provides a 46.7 recovery and a purification factor of 12.3,whereas when the adsorbent is treated In a similar manner to Example VI,the purification with an alkali metal trimetaphosphate the recovery isone of influenza virus A/Jap. 305 is carried out on 300 g. of hundredand one percent obtained at a purification factor alkali and TMP treatedcalcium dihydrogen orthophosof 31.

phate monohydrate adsorbent. The virus is first adjusted Similar resultsare obtained when alkali treated calcium to a pH of 9.0. The followingresults were obtained: dihydrogen orthophosphate monohydrate adsorbentsof Lowry Volume, protein, G OA/mg. Percent Sample ml. CCA mg. L.P. P.F.recovery Infected allantoic fluid 20, 000 542, 000 5, 550 07. 7 Columnfraction 1. 21, 000 102,900 5, 040 20. 4 Column fraction 2 240 001, 584140. 4 4100. 47. e 111 By the above procedure, a recovery of 111 percentExamples II and III are employed to purify the influenza of the virus isobtained at a purification factor of 47.6. virus strains reported in theprior example.

EXAMPLE VIII 7 EXAMPLE X In a manner similar to the Examples VI andVII,- a- Two columns (columns A and B) are prepared: for each, series ofruns are carried out on a variety of influenza 100 grams of CA(I-I PO -HO are prepared accordvirus strains, identified below according to theconditions ing to Example IV and placed in a glass column. To each andwith the alkali and TMP treated adsorbent of the column there is thendelivered one liter of undiluted inpresent invention. The columns usedhave varying capacifected allantoic fluid containing A/Jap. 170. viruswhich ties of from 46 to 102.1. The following table summarizes arepreviously adjusted with 0.01 M sodium trimetaphosthe results obtainedwith these various strains of influenza: phate. For column A the fluidis pre-heated to 37 C.

Total CCA Percent CCA/mg. Run No. Strain recovered recovery RF. LP.

79 43 4028 101 31 3403 87 23 5230 07 32 3170 13 B[Maryland 5, 003, s0080 38' 4, 500 12 A/Jap 170 0,711,300 102 25 1,001

Based on specific activity before and after purification S.A.:CCA/mg.Lowry Protein.

By the above procedure, recovery of the various strains while for columnB the fluid is cooled to 10 C. prior to of influenza viruses varied from67 percent to 102 percent application to the column. For both columns,recoveries at purification factors from 25 to 43. by elution with 1 M pH6.8 phosphate buffer are as fol- Similar results are obtained when thealkali and TMP lows: treated adsorbents of Example V are employed.

EXAMPLE IX Recovery,

When the alkali treated calcium dihydrogen orthophos- Sample VolumeTotal CCA Percent phate monohydrate adsorbent of Example I is employedInfected fluid 1,000 273,000 to purify a variety of influenza virusstrains, the proceifig 1 1,210 4840 7 V dure of the prior example isemployed, except that the FractionZ 23 03, 311 34.2 adsorbent and virusinfected allantoic fluids are not treated fifig 1 1 250 5 O00 2 with analkali metal trimetaphosphate. The results there- Fraction 2 :I 27 113:202 44.5

of are set forth below:

mp 305 Ann Arbor B/GL It should be noted that in both columns aboveimprove- =1 Vlms ment in the concentration of CCA units per ml. is 1g ggggg 2g 33?, 38% 13 333 achieved from the starting level of 273 CCA perml. in Percent iactor 40.7 03.0 20.4 14.1 the allantoic fluid. Theconcentration is approximately Purifiwfimfecmfl m1 15-fold for bothcolumns 4057 CCA per ml. in A and *Infected allantoic fluid. 4196 CCA.per ml. in B).

7 EXAMPLE XI In a manner comparable to the preceding example, 750 ml. ofAZ/Iap. 170 virus are applied to the column at a temperature of 4 C.after having been diluted with an equal volume of water and thenadjusted to 0.01 M sodiurn trimetaphospliate. The recovery of virus soprocessed is as follows:

' Recovery, Sample Volume Total A. percent Infected Fluid 1, 500 64, 500Fraction 1 1, 680 6, 720 1. 7 Fraction 2 29 40, 136 62 EXAMPLE XII In amanner similar to the preceding examples, a column is prepared in whichcold 3 N sodium hydroxide is added dropwise to the adsorbent until a pHof 3.0 is reached. The recovery of A/Iap. 170 virus so processed is asfollows:

Volume, Total Percent Sample ml. CCA/ml. 0 CA Recovery InfectedAllantoic fluitL 750 224 168, 000 Column Fraction 1 1, 690 4 6, 760 4. 0Column Fraction 2".-- 68 1, 446 98, 328 58. 5

EXAMPLE XIII Two columns are prepared: for each, 100 grams of Ca(H PO -HO are prepared according to Example IV. After adjusting to pH 8.0 with0.01 molar sodium trimetaphosphate buffer Wash, the adsorbents aremaintained in the bufifer wash overnight at room temperature. Theadsorbents are then placed in glass columns and ad- Example IV, andplaced in a glass column. The adsorbent is then Washed with 0.01 molarpH 10.0 sodium trimetaphosphate to a column pH of 9.0. To the column,there is then delivered 750 ml. of diluted B/Maryland strain infectedallantoic fluid, adjusted to pH 8 with a as that obtained at a TMPcolumn pH of 7.0 and 7.5 respectively, as shown in Example XIII.However, some improvement in the concentration of CCA units per ml. isachieved with a trimetaphosphate treated column having a pH of 9.0.

EXAMPLE XV In a manner similar to Example IV, the purification ofB/Maryland strain of influenza virus is carried out on four columnscontaining 100 grams of Ca(H PO -H O per column. The virus is elutedwith 1.0 molar phosphate buffer, pH 6.8, 7.5, 8.0 and 9.0 respectively.The following results are obtained:

Elution Volume, Total Percent Sample pH ml. CCA/ml GOA recovery InfectedAllantoic Fluid 500 427 213, 500 Column A- 6. 8 38 4, 595 174, 610 82Column B- 7. 5 42 5, 153 216, 426 101 Column 0. 8. 0 42 5, 302 222, 624104 Column D- 9. 0 53 4, 782 253, 446 119 justed to pH 7.0 (column A)and pH 7.5 (column B) with 0.01 molar sodium trimetaphosphate bufferwash. A volume of 500 ml. of B/Maryland strain infected allantoic fluidis applied to each column, after having been diluted with an equalvolume of distilled water and adjusted to pH 8.0 with a 0.01 molarsodium trimetaphosphate solution. The virus is recovered from thecolumns EXAMPLE XVI In a manner similar to Example XV, the purificationof A /AA strain of influenza virus is carried out on four columns. Thevirus is eluted with 1.0 molar phosphate buffer, pH 6.8, 7.5, 8.0 and9.0. The following results were obtained:

Elution Volume, Total Percent; Smple pH ml. 0 CA/ml. 0 CA recoveryInfected Allantoic fluid 750 201 150, 750 C umn A i- 8 21 5, 054 106,134 71 Column B 7. 5 38 2, 624 99, 712 66 Column C 8. 0 43 3, 258 140,094 93 Column D 9. 0 37 2, 479 91, 723 61 by elution with 1.0 molarphosphate buffer. The following results are obtained:

EXAMPLE XVII In a manner similar to the preceding examples, the puri-Sample 3 gg g fication of A/PR-8 strain of influenza virus is carriedout I f t d H fl d 00 91 198 500 on four columns. The virus is elutedwith 1.0 molar phosnece A antoic ui 5 -7 Column A Fraction 67 1, 469 98'423 91 phate buffer, 6.8, 7.5, 8.0 and 9.0. The following Column B,Fraction 2.. 70 1, 204. 84, 280 77 results are obtamed:

Elution Volume, otal Percent Sample pH ml. OCA/ml. GOA recovery InfectedAllantoic fluids- 750 329 246, 750

A 34 7, 130 242, 420 as EXAMPLE XIV g. of Ca(H PO -H O are preparedaccording to EXAMPLE XVIII In a manner similar to the precedingexamples, the

purification of A/Jap. 170 strain of influenza virus is carried out oneight columns. The virus is eluted with 1.0 molar phosphate buffer at pHranges from 5.0 to 9.0. The following results are obtained:

from about one to not more than about five parts of water with analkali, at a temperature of not more than about 40 C., to adjust the pHof the suspension to a range of from about 5.5 to about 8.0;

Elution Volume, Total Percent Sample pH ml. CCA/ml. GOA recoveryInfected Allantoie fluid 750 124 93, 000 Column A 5. s 32 2,104 67, 32872 7. 5 33 1, 742 57, 435 02 3. 33 1, 375 45, 375 49 0. 0 50 s26 48, 73452 750 232 174,000 5. 0 30 3, 705 111,150 64 5. 34 4, 728 160, 752 02 5.0 39 4, 660 131, 740 104 0. 5 36 4,385 175, 350 101 EXAMPLE XIX (b)washing the Solid of said suspenslon with water unt1l the water wash hasa pH at least about 0.2 unit In a manner similar to the precedingexamples, the higher than the adjusted pH of the above suspensionpurification of A /Taiwan strain of influenza virus is and is in therange of from about 5.7 to about 8.2; carried out on six columns. Thevirus is eluted with 1.0 and molar phosphate bufler at pH ranges from5.0 to 8.0. (c) washlng the solid of said washed suspension with Thefollowing results are obtained: an aqueous alkali metal trimetaphosphatesolution Elution Volume, Total Percent Sample ml. CCA/ml. GOA recoveryInfected Allantoic fluid 750 295 221, 250 A e. 0 40 4, 933 197, 520 6. 534 4, 855 105, 070 74 7. 5 33 4, 137 157, 206 71 s. 0 43 4, 200 159, 00072 750 206 154,500 5. 5 40 2, 831 130, 226 34 5. 0 3s 2, 301 00, 853 59EXAMPLE XX until the pH of the suspension is in the range of from about7.5 to about 8.5. In a manner similar to the preceding examples, the 35purification of A/Jap. 170 strain of influenza virus is carried out on aseries of columns. The undiluted virus fluid is applied to the columnsand is eluted with phosphate buffer, pH 6.8 and 8.0 at 0.5 M, 0.75 M,and 1.0 M

3. A method of preparing a stable adsorbent particularly adapted for thepurification of virus which comprises, suspending calcium dihydrogenorthophosphate monohydrate with water in an amount of from about oneconcentrations. Recoveries of the virus so processed are to not morethan about five Parts Of Water P P of as follows: the compound,maintalnrng the suspension at a tempera- Elution pH and eoncen- Volume,0 CA Total Percent Sample tration ml. ml. COA recovery InfectedAllantoic fluid 500 231 115, 500 A 6. 8 33 2, 930 96, 690 84 8. 0 38 2,799 106, 362 92 6. 8 39 2,718 101,302 82 ,8. 0 42 2, 750 115, 500 100 6.8 28 3, 400 95,200 82 Column F- 8. 0 25 2, 963 73, 875 64 While theforegoing invention has been described with some degree of particularityin the descriptive matter and in the specific examples provided, thesame was for purposes of clarity and definition and is not to beconsidered in any way as a limit on the scope of the invention. Theinvention is to be limited only by the claims appended hereto.

What is claimed is:

1. A virus adsorbent which comprises the product produced by:

(a) treating an aqueous suspension of calcium dihydrogen orthophosphatemonohydrate containing from about one to not more than about five partsof water with an alkali, at a temperature of not more than about 40 C.,to adjust the pH of the suspension to a range of from about 5.5 to about8.0;

(b) washing the solid of said suspension with water until the water washhas a pH at least about 0.2 unit higher than the adjusted pH of theabove suspension and is in the range of from about 5.7 to about 8.2.

2. A virus adsorbent which comprises the product produced by:

(a) treating an aqueous suspension of calcium dihydrogen orthophosphatemonohydrate containing ture of not more than about 40 C., addingsuflicient alkali to said suspension to adjust the pH thereof to therange of from about 5.5 to about 8.0, decanting excess water from themixture, and washing the adsorbent with water until the water wash has apH at least about 0.2 unit higher than the adjusted pH of the abovesuspension and is in the range of about 5.7 to about 8.2.

4. A method as described in claim 3 for preparing a stable adsorbent forvirus purification which comprises suspending calcium dihydrogenorthophosphate monohydrate with water in the ratio of from about one toabout two parts of water per part of the salt, maintaining thesuspension at a temperature of not more than about 40 C., slowly addingto said suspension with mixing suflicient alkali to adjust the pH of themixture to about 6.5, decanting excess water from the mixture andwashing the adsorbent with water until the remaining supernatant liquidhas a pH of about 7.0.

5. A method as described in claim 3 for preparing a stable activatedadsorbent for virus purification which comprises suspending calciumdihydrogen orthophosphate monohydrate with water in the ratio of onepart of the salt to about five parts of water, maintaining saidsuspension at a temperature of not more than about 40 C.,

adding to said suspension sufficient alkali to adjust the pH thereof tothe range of from about 5.5 to about 8.0, decanting excess water fromthe mixture, washing the mixture with Water until the water Wash has apH at least about 0.2 unit higher than the adjusted pH of the abovesuspension and has a pH of at least about 5.7 and not more than about8.2, and further washing said suspended adsorbent with a solution of analkali metal trimetaphosphate until the suspension has a pH of about 7.5to about 8.5.

6. A method as described in claim 3 for preparing a stable activatedadsorbent for virus purification which comprises suspending calciumdihydrogen orthophosphate monohydrate with water in the ratio of aboutone part of the salt to about five parts of Water, maintaining thesuspension at a temperature of not more than about 40 0., adding to saidsuspension sufiicient alkali to adjust the pH thereof to the range offrom about 5.5 to about 8.0, decanting excess Water from the mixture,Washing the mixture with Water until the Water Wash has a pH at leastabout 0.2 unit higher than the adjusted pH of the above suspension andhas a pH of about 5.7 to about 8.2 and further washing said suspendedadsorbent with an alkali metal trimetaphosphate solution until thesuspension has a pH of about 8.0.

7. A method as described in claim 3 for preparing a stable activatedadsorbent for virus purification which comprises suspending calciumdihydrogen orthophosphate monohydrate with water in the ratio of aboutone part of salt to about two parts of Water, maintaining saidsuspension at about room temperature, adding suflicient alkali to thesuspension to bring the pH to about 6.5, decanting excess water from themixture, washing the mixture with Water until the Water Wash has a pH ofabout 6.7 to about 7.5 and further Washing said suspended adsorbent witha sodium trimetaphosphate solution until the suspension has a pH ofabout 8.0.

References Cited UNITED STATES PATENTS 7/1965 Hennessen et al 231099/1968 Saeman 23109 OTHER REFERENCES PATRICK P. GARVIN, Primary ExaminerU.S. Cl. X.R. 23109; 424-89

